The laboratory has been focused during the last year on immune-mediated tumor cell destruction. Natural killer (NK) cells and cytotoxic T cells (CTL) lyse virally-infected and tumor cells by 2 main mechanisms:(1) the exocytosis of lytic granules and (2) the expression of cytotoxic molecules like tumor necrosis factor (TNF) or related molecules like Fas-ligand. We have investigated the importance of these mechanisms in cytotoxic immune responses to a murine renal cancer Renca. We successfully isolated CTL specific for the Renca tumor. These CTL exclusively used FasL or perforin/granzymes to lyse Renca cells in vitro. Furthermore,the CTL showed some therapeutic efficacy when transfered to tumor-bearing mice in vivo. Interestingly CTL derived from perforin "knockout" mice could efficiently destroy pulmonary metastases of Renca. This suggests that the perforin pathway was not essential for antitumor effects of the CTL, at least in this metastasis model. We are currently investigating mechanisms underlying how metastases of this tumor in the lungs and various other anatomical sites (such as the peritoneum or liver) are eliminated by CTL. The cytolytic granules of cytotoxic lymphocytes contain a variety of unique proteins, including a pore-forming protein (perforin) and a family of serine protease enzymes called granzymes. We have further studied the expression of granzyme M (met-ase), a novel granzyme that we had previously cloned. Using a monoclonal antibody specific for granzyme M we have detected this granzyme in freshly isolated NK cells, NKT cells and T gamma delta cells by western blotting and immunohistochemistry. T alpha beta cells do not seem to express this granzyme even after activation. The expression pattern of Granzyme M contrasts with that of the other granzymes and perforin, which can be easily detected in activated T alpha beta cells. This suggests that Granzyme M may be involved in innate rather than adaptive immunity. We are trying to define the biological role of this enzyme, and have obtained mice where granzyme M has been ablated by gene targeting.